Over the past five years we have investigated the biochemical and molecular aspects of hK2 and its proform (phK2) and have developed highly sensitive and specific analytic assays for these biomarkers. Our studies have shown that these proteins are localized predominately in the prostate, are hormonally regulated and have potential proteolytic role in the activation of PSA from proPSA. Preliminary studies also have shown that the ratio of serum concentration of hK2 to free-PSA is useful for identifying those patients with borderline elevated PSA concentrations who are most likely to have prostate cancer. Specific Aim 1 will employ the proven molecular, biochemical and analytic techniques used to characterize and measure hK2 to further characterize and develop assays for three new prostate cancer markers: inhibitor - 6 complex (hK2-PI6), hK2 variant (hK2-v) and PSA variant (PSA-v). Vectors will be developed to produce recombinant forms of these proteins and specific monoclonal antibodies will be produced. These antibodies will be used for purification of endogenous proteins, immunohistochemistry studies and development of sensitive and specific immunoassays. Specific Aim 2 involves a collaboration with clinical colleagues in Urology, Oncology and the Cancer Center. Blood, tissue and clinical follow-up information will be collected for three large cohorts of patients. One cohort will include 3000 men undergoing transrectal needle biopsy of the prostate. Another cohort will include 300 men with rising PSA concentrations and negative needle biopsy undergoing saturation biopsy of the prostate under anesthesia. A third cohort will include 240 men with advanced hormone refractory prostate cancer. Each of these cohorts will have PSA, free PSA, hK2 and phK2 measured in their blood to determine the utility of these markers for improving cancer detection and predicting outcomes. In addition, cancer prediction algorithms will be validated with blood from 600 Asian men from Korea undergoing prostate biopsy. Aliquots of blood from the cohorts also will be held frozen to evaluate the utility of hK2-PI6, hK2-v and PSA-v once assays have been developed. The major goal of these studies will be the development of the most practical and accurate assays to improve the detection, diagnosis and management of prostate cancer.